Probiotic composition based on the enterococcus strain and used as a treatment means and method for the production thereof

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may contain, in addition to the active component, colorants, flavors, stabilizers, buffers, artificial and natural sweeteners, dispersants, thickeners, solubilizing agents, and the like.

[0065] Exemplary pharmaceutically carriers include sterile aqueous of non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable or seed oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. A composition of inactivated probiotic bacteria may also be lyophilized using means well known in the art, for subsequent reconstitution and use according to the invention. Also of interest are formulations for liposomal delivery, and formulations comprising encapsulated or microencapsulated inactivated probiotic bacteria.
[0066] The formulations of the present invention may also include known antioxidants, buffering agents, and other agents such as coloring agents, flavorings, vitamins or minerals.
For example, a subject formulation may also contain one or more of the following minerals: calcium citrate (15-350 mg) ; potassium gluconate (5-150 mg); magnesium citrate (5-15 mg) ; and chromium picollinate (5-200 gag). In addition, a variety of salts may be utilized, including calcium citrate, potassium gluconate, magnesium citrate and chromium picollinate. Thickening agents may be added to the compositions such as polyvinylpyrrolidone, polyethylene glycol or carboxymethylcellulose. Exemplary additional components of a subject formulation include assorted colorings or flavorings, vitamins, fiber, milk, fruit juices, enzymes and other nutrients.
Exemplary sources of fiber include any of a variety of sources of fiber including, but not limited to: psyllium, rice bran, oat bran, corn bran, wheat bran, fruit fiber and the like. Dietary or supplementary enzymes such as lactase, amylase, glucanase, catalase, and the like can also be included. Chemicals used in the present compositions can be obtained from a variety of commercial sources, including, e. g., Spectrum Quality Products, Inc (Gardena, Calif. ), Sigma

Chemicals (St. Louis, Mo. ), Seltzer Chemicals, Inc., (Carlsbad, Calif. ) and Jarchem Industries,

Inc. , (Newark, N. J. ).
[0067] A subject formulation may also include a variety of carriers and/or binders. An exemplary carrier is micro-crystalline cellulose (MCC) added in an amount sufficient to complete dosage total weight. Carriers can be solid-based dry materials for formulations in tablet, capsule or powdered form, and can be liquid or gel-based materials for formulations in liquid or gel forms, which forms depend, in part, upon the routes of administration.
[0068] Typical carriers for dry formulations include, but are not limited to: trehalose, malto- dextrin, rice flour, micro-crystalline cellulose (MCC) magnesium sterate, inositol, fructo-

oligosaccharide (FOS), gluco-oligosaccharide (GOS), dextrose, sucrose, and like carriers.

Where the composition is dry and includes evaporated oils that produce a tendency for the composition to cake (adherence of the component spores, salts, powders and oils), dry fillers which distribute the components and prevent caking are included. Exemplary anti-caking agents include MCC, talc, diatomaceous earth, amorphous silica and the like, and are typically added in an amount of from approximately 1% to 95% by weight. It should also be noted that dry formulations which are subsequently rehydrated (e. g. , liquid formula) or given in the dry state (e. g. , chewable wafers, pellets, capsules, or tablets) can be used instead of initially hydrated formulations. Dry formulations (e. g. , powders) may be added to supplement commercially available foods (e. g. , liquid formulas, strained foods, or drinking water supplies).
Similarly, the specific type of formulation depends upon the route of administration.
[0069] Suitable liquid or gel-based carriers include but are not limited to: water and physiological salt solutions; urea; alcohols and derivatives (e. g. , methanol, ethanol, propanol, butanol); glycols (e. g. , ethylene glycol, propylene glycol, and the like). Generally, water-based carriers possess a neutral pH value (e. g. , pH 7.0 1.0 or 0.5 pH units). The compositions may also include natural or synthetic flavorings and food-quality coloring agents, all of which must be compatible with maintaining viability of the lactic acid-producing microorganism. Well- known thickening agents may also be added to the compositions such as corn starch, guar gum, xanthan gum, and the like.
[0070] Preservatives may also be included within the carrier including methylparaben, propylparaben, benzyl alcohol and ethylene diamine tetraacetate salts. Well-known flavorings and/or colorants may also be included within the carrier. The compositions of the present invention may also include a plasticizer such as glycerol or polyethylene glycol (e. g. , in a molecular weight range of MW=800 to 20,000). The composition of the carrier can be varied so long as it does not interfere significantly with the pharmacological activity of the inactivated probiotic bacteria.
[0071] Inactivated probiotic bacteria can be formulated to be suitable for oral administration in a variety of ways, for example in a liquid, a powdered food supplement, a paste, a gel, a solid food, a packaged food, a wafer, a tablet, a lozenge, a capsule, and the like. Other formulations will be readily apparent to one skilled in the art.
[0072] In general, the pharmaceutical compositions can be prepared in various forms, such as granules, tablets, lozenges, pills, suppositories, capsules (e. g. adapted for oral delivery), microbeads, microspheres, liposomes, suspensions, and the like. The inactivated probiotic

bacteria useful in the invention can be prepared in a variety of formulations, including conventional pharmaceutically acceptable carriers, and, for example.

[0073] Inactivated probiotic bacteria may be formulated with an inert diluent or with an assimilable edible carrier, or may be enclosed in hard or soft shell gelatin capsule, or may be compressed into tablets designed to pass through the stomach (i. e. , enteric coated), or may be incorporated directly with the food of the diet. For oral therapeutic administration, the inactivated probiotic bacteria may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
[0074] The tablets, troches, pills, capsules, and the like, as described above, may also contain the following: a binder such as gum tragacanth, acacia, a starch (such as corn starch), or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid, and the like ; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, lactose or saccharin may be added or a flavoring agent such as peppermint, oil or wintergreen or cherry flavoring. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present as coatings or to otherwise modify the physical form of the dosage unit. For instance, tablets, lozenges, pills or capsules or bacteria in suspension may be coated with shellac, sugar or both.
[0075] A syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. In addition, the inactivated probiotic bacteria may be incorporated into sustained-release preparations and formulations.
[0076] Pharmaceutical grade organic or inorganic carriers and/or diluents suitable for oral use can be used to make up compositions comprising the therapeutically-active compounds.
Diluents known to the art include aqueous media, vegetable and animal oils and fats.
Stabilizing agents, wetting and emulsifying agents, salts for varying the osmotic pressure or buffers for securing an adequate pH value, can also be added.
[0077] Some other examples of substances which can serve as pharmaceutical carriers are sugars, such as lactose, glucose and sucrose; starches such as corn starch and potato starch ; cellulose and its derivatives such as sodium carboxymethycellulose, ethylcellulose and cellulose acetates; powdered tragancanth; malt; gelatin; talc; stearic acids; magnesium stearate;

calcium sulfate; calcium carbonate; vegetable oils, such as peanut oils, cotton seed oil, sesame oil, olive oil, corn oil and oil of theobroma ; polyols such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; agar; alginic acids ; pyrogen-free water; isotonic saline; cranberry. extracts and phosphate buffer solution; skim milk powder; as well as other non-toxic compatible substances used in pharmaceutical formulations such as Vitamin C, estrogen and echinacea, for example. Wetting agents and lubricants such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, lubricants, excipients, tabletting agents, stabilizers, anti-oxidants and preservatives, can also be present.

[0078] A colloidal dispersion system may be used for oral delivery of inactivated probiotic bacteria. Colloidal dispersion systems include macromolecule complexes, nanocapsules, microspheres, beads, liposomes and the like.
[0079] The inactivated probiotic bacteria of the present invention may be formulated for administration as suppositories. A low melting wax, such as a mixture of fatty acid glycerides or cocoa butter is first melted and the inactivated probiotic bacteria are dispersed homogeneously, for example, by stirring. The molten homogeneous mixture is then poured into conveniently sized molds, allowed to cool, and to solidify.
[0080] The inactivated probiotic bacteria of the present invention may be formulated for vaginal administration. Pessaries, tampons, creams, gels, pastes, foams or sprays, may contain agents in addition to the bacteria, such carriers, known in the art to be appropriate.
[0081] In some embodiments, inactivated probiotic bacteria are formulated for delivery by inhalation. As used herein, the term"aerosol"is used in its conventional sense as referring to very fine liquid or solid particles carries by a propellant gas under pressure to a site of therapeutic application. The term"liquid formulation for delivery to respiratory tissue"and the like, as used herein, describe compositions comprising inactivated probiotic bacteria with a pharmaceutically acceptable carrier in flowable liquid form. Such formulations, when used for delivery to a respiratory tissue, are generally solutions, e. g. aqueous solutions, ethanolic solutions, aqueous/ethanolic solutions, saline solutions and colloidal suspensions.
[00$2] In general, aerosolized particles for respiratory delivery must have a diameter of 12 microns or less. However, the preferred particle size varies with the site targeted (e. g, delivery targeted to the bronchi, bronchia, bronchioles, alveoli, or circulatory system). For example, topical lung treatment can be accomplished with particles having a diameter in the range of 1.0 to 12.0 microns. Effective systemic treatment requires particles having a smaller diameter, generally in the range of 0.5 to 6.0 microns. Thus, in some embodiments, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about
90%, or more, of an aerosolized formulation comprising inactivated probiotic bacteria for delivery to a respiratory tissue is composed of particles in the range of from about 0.5 to about

12 micrometers, from about 0.5 to about 6 micrometers, or from about 1.0 to about 12 micrometers.

[0083] The formulation for delivery to a respiratory tissue may be provided in a container suitable for delivery of aerosolized formulations. U. S. Patents 5,544, 646 ; 5,709, 202;

5,497, 763 ; 5,544, 646; 5, 718, 222; 5,660, 166; 5,823, 178; 5,829, 435 ; and 5,906, 202 describe devices and methods useful in the generation of aerosols suitable for drug delivery, any of which can be used in the present invention for delivering a formulation comprising inactivated probiotic bacteria to a respiratory tissue. In some embodiments, the invention provides a container, which may be a disposable container, having at least one wall that is collapsible or movable upon application of a force, wherein at least one wall has an opening. A porous membrane having pores in a range of from about 0.25 microns to about 6 microns covers the opening. The container comprises a flowable liquid formulation comprising inactivated probiotic bacteria. Upon application of a force, the flowable liquid formulation is forced through the pores in the membrane and is aerosolized. The container may be provided in any known configuration, e. g. , a blister pack. The container may be provided together with an aerosol delivery device, such that the aerosolized formulation exits the container and proceeds through a channel in an aerosol delivery device and into the respiratory tract of an individual.

[0084] When a pharmaceutical aerosol is employed in this invention, the aerosol contains inactivated probiotic bacteria, which can be dissolved, suspended, or emulsified in a mixture of a fluid carrier and a propellant. The aerosol can be in the form of a solution, suspension, emulsion, powder, or semi-solid preparation. Aerosols employed in the present invention are intended for administration as fine, solid particles or as liquid mists via the respiratory tract of a patient. Various types of propellants known to one of skill in the art can be utilized.
Examples of suitable propellants include, but is not limited to, hydrocarbons or other suitable gas. In the case of the pressurized aerosol, the dosage unit may be determined by providing a value to deliver a metered amount.
[OOS5] Administration of formulation comprising inactivated probiotic bacteria can also be carried out with a nebulizer, which is an instrument that generates very fine liquid particles of substantially uniform size in a gas. Preferably, a liquid containing the inactivated probiotic bacteria is dispersed as droplets. The small droplets can be carried by a current of air through an outlet tube of the nebulizer. The resulting mist penetrates into the respiratory tract of the patient.

[0086] A powder composition containing inactivated probiotic bacteria, with or without a lubricant, carrier, or propellant, can be administered to a mammal in need of therapy. This embodiment of the invention can be carried out with a conventional device for administering a powder pharmaceutical composition by inhalation. For example, a powder mixture of the compound and a suitable powder base such as lactose or starch may be presented in unit dosage form in for example capsular or cartridges, e. g. gelatin, or blister packs, from which the powder may be administered with the aid of an inhaler.

[0087] Although the compositions of the invention may be directly ingested, inhaled, or otherwise administered, or used as an additive in conjunction with foods, it will be appreciated that they may be incorporated into a variety of foods and beverages. The terms"food,""food product,"and"foodstuff"are used interchangeably herein and include, in addition to foods commonly consumed by humans and domesticated animals, functional foods, pharmafoods, designer foods, and nutraceuticals. Suitable foods and beverages include, but are not limited to, yogurts, ice creams, cheeses, baked products such as bread, biscuits and cakes, dairy and dairy substitute foods, soy-based food products, grain-based food products, starch-based food products, confectionery products, edible oil compositions, spreads, breakfast cereals, infant formulas, juices, power drinks, and the like. Within the scope of the term"foods"are to be included in particular food likely to be classified as functional foods, i. e. "foods that are similar in appearance to conventional foods and are intended to be consumed as part of a normal diet, but have been modified to physiological roles beyond the provision of simple nutrient requirements" (NFA Policy Discussion Paper 7/94).
[0088] As non-limiting examples, subject inactivated probiotic bacteria are in some embodiments incorporated into milk products, including liquid, solid, semi-solid, and powdered milk products. Thus, the invention provides a milk-based food product comprising subject inactivated probiotic bacteria. A subject food product includes milk, and any food products made from or containing milk, including, but not limited to, cheese, yogurt, butter, ice cream, and other frozen desserts, whipped toppings, cream, custard, pudding, nutritional drinks, infant formula, and milk chocolate. In some embodiments, the invention provides a food product comprising subject inactivated probiotic bacteria, where the food product is a milk product, and where the milk product is any of powdered infant formula ; liquid infant formula; liquid milk; powdered milk; a flavored liquid milk; a flavored powdered milk; yogurt; a yogurt-based beverage; cheese; butter; cream; and the like; or combinations of the foregoing.
A subject milk-based food product includes any food product that includes milk as a component, or that is made using milk.

[0089] Cheeses include any fresh or ripened cheese. Such cheese include, but are not limited to, Campesino, Chester, Danbo, Drabant, Herregard, Manchego, Provolone, Saint Paulin, Soft cheese, Taleggio, White cheese, Cheddar, Colby, Edam, Muenster, Gruyere, Emmenthal,

Camembert, Parmesan, Romano, Mozzarella, Feta; cottage cheese; cream cheese, Neufchatel, etc.
[0090] In some embodiments, the milk-based food product is a processed cheese food product.
Processed cheese food products include, but are not limited to, pizza, ready-to-eat dishes, toast, burgers, lasagna, dressing, sauces, cheese powder, cheese flavor, and processed cheese.
[0091] Subject inactivated probiotic bacteria are in some embodiments formulated with nutritional beverages, e. g. , peptide-based preparations; beverages comprising nutrients that are easily absorbed by the gut epithelium, e. g. , peptides, fatty acids, electrolytes, monosaccharides, disaccharides, and the like ; nutritional beverages such as Ensured ; and the like.
[0092] In some embodiments, subject inactivated probiotic bacteria are incorporated into soy- based food products, including liquid, solid, semi-solid, and powdered soy food products.
Thus, the invention provides a soy-based food product comprising subject inactivated probiotic bacteria. Soy-based food products include, but are not limited to, soy infant formula, soy "milk,"soy-based food bars, and the like. See, e. g. , U. S. Patent Publication Nos. 20030219526 and 20030054087.
[0093] In some embodiments, subject inactivated probiotic bacteria are incorporated into grain-based food products, include food products that comprise whole grains, food products that comprise processed grains (e. g. , milled grains, such as flour; parboiled grains ; puffed grains; grains processed for breakfast cereals; and the like). Grain-based food products include those made using wheat, rice, oats, barley, rye, corn, amaranth, flax, millet, sorghum, triticale, or a combination of two or more grains. In some embodiments, subject inactivated probiotic bacteria are incorporated into flour-based food products, including breads, cookies, cakes, pastas, etc., made with milled grain (s). In some embodiments, subject inactivated probiotic bacteria are incorporated into gluten-free food products (e. g., food products free of wheat, rye and barley, or any of their derivatives), wheat-free food products, and casein-free food products.
[0094] In some embodiments, subject inactivated probiotic bacteria are incorporated into starch-based food products, e. g. , products made using potato starch.

Additional agents [0095] Inactivated probiotic bacteria can be formulated with additional agents, which agents may be inert or active agents. For example, preservatives and other additives may also be present such as, for example, antimicrobial agents (e. g. , antibacterials, antivirals, antifungals, etc. ), antioxidants, chelating agents, and inert gases and the like. In some embodiments, inactivated probiotic bacteria are formulated with one or more additional therapeutic agent for the treatment of gastrointestinal inflammation, diarrhea, irritable bowel syndrome, microbial infection, allergy, etc.

[0096] Inactivated probiotic bacteria can be combined with conventional agents used for treatment of gastrointestinal inflammation, where appropriate. Exemplary agents used in conventional gastrointestinal inflammation therapy, such as those used in therapy for chronic gastrointestinal inflammation such as in IBD, include, but are not necessarily limited to, 5- aminosalicylate (5-ASA), sulfasalazine, corticosteroids, azathioprine, cyclosporine, and methotrexate, as well as tumor necrosis factor-a (TNF-a) antagonists, cytokines such as IL-10, or other drug useful. in the treatment of chronic gastrointestinal inflammation.
[0097] Such additional agents can be administered separately or included in the inactivated probiotic bacteria composition. In addition, inactivated probiotic bacteria can be formulated with other agents, e. g. , anti-inflammatory agents, with the proviso that such agents do not substantially interfere with the efficacy of inactivated probiotic bacteria. Exemplary agents include, but are not necessarily limited to, antacids, H2 blockers, proton pump inhibitors, and the like (e. g., famotidine, ranitidine hydrochloride, omeprazol, and the like). Suitable H2 blockers (histamine type 2 receptor antagonists) include, but are not limited to, Cimetidine (e. g. , Tagamet, Peptol, Nu-cimet, apo-cimetidine, non-cimetidine); Ranitidine (e. g. , Zantac,

Nu-ranit, Novo-randine, and apo-ranitidine); and Famotidine (Pepcid, Apo-Famotidine, and Novo-Famotidine).

[009$1 Subject inactivated probiotic bacteria can be formulated together with an immunosuppressive agent. Suitable immunosuppressive agents include, but are not limited to, a steroidal immunosuppressive agent, azathioprine, 6-mercaptopurine, methotrexate, cyclosporine, tacrolimus, mycophenolate mofetil, thalidomide, and the like.
[0099] Suitable TNF-a antagonists that can be formulated with a subject inactivated probiotic formulation include soluble TNF-a receptors, chimeric TNF-a receptors, antibodies to TNF-a, etc. Suitable TNF-a antagonists include, but are not limited to, ENBREL (D (a dimeric fusion protein consisting of the extracellular ligand-binding portion of the human 75 kilodalton (p75)

TNFR linked to the Fc portion of human IgGl ; Smith et al. (1990) Science 248 : 1019-1023 ;

Mohler et al. (1993) J Immunol. 151: 1548-1561; U. S. Pat. No. 5,395, 760; and U. S. Pat. No.
5,605, 690); Infliximab (REMICADE; ; a chimeric monoclonal anti-TNF-a antibody that includes about 25% mouse amino acid sequence and about 75% human amino acid sequence; Elliott et al. (1993) Arthritis Rheum. 36: 1681-1690; Elliott et al. (1994) Lancet 344 : 1105-1110;

Baert et al. (1999) Gastf oenterology 116: 22-28); and Adalimumab (HUMIRATM ; a human, full-length IgGl monoclonal antibody that was identified using phage display technology.

Piascik (2003) J. Am. Pharm. Assoc. 43: 327-328); and the like.
[00100] Inactivated probiotic bacteria can be combined (e. g. , formulated with) with conventional agents that treat diarrhea, e. g. , loperamide (Imodium (M, hnodium (M A-D); bismuth subsalicylate; diphenyloxylate/atropine (Lomotil (g)) ; attapulgite (Kaopectate (g)) ; and the like.
[00101] Inactivated probiotic bacteria can be formulated with one or more antibiotics. Because the inactivated probiotic bacteria of the instant invention are non-viable or have reduced viability, an antibiotic can be included in the formulation without concern about adverse effects on probiotic viability. Antibiotics include, but are not limited to, Gentamicin; Vancomycin ; Oxacillin ; Tetracyclines; Nitroflurantoin; Chloramphenicol; Clindamycin; Trimethoprim- sulfamethoxasole; a member of the Cephlosporin antibiotic family (e. g. , Cefaclor, Cefadroxil,

Cefixime, Cefprozil, Ceftriaxone, Cefuroxime, Cephalexin, Loracarbef, and the like); a member of the Penicillin family of antibiotics (e. g. , Ampicillin, Amoxicillin/Clavulanate,

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